Download Affecting Cucurbit hormonal status by a ZYMV transient expression

Affecting Cucurbit hormonal status by a ZYMV transient expression
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V. Davidovich1, D. Leibman2, A. Gal-On2, and R. Perl-Treves1*
1
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan
52900, Israel
2
Department of Plant Protection, Agricultural Research Organization, Bet Dagan 50250, Israel
*
Corresponding author e-mail: [email protected]
Keywords: Zucchini yellow mosaic virus, GA20 oxidase, ACC synthase
Abstract
Squash and cucumber plants were infected with ZYMV strains engineered to
over-express enzymes involved in ethylene and gibberellin biosynthesis. Presence of
the inserted ACS gene within the viral RNA was transient and the insert was deleted
after 20-40 days. Ethylene over-expressing plants produced very high ethylene levels
and were severely stunted. Monoecious cucumbers produced earlier, consecutive
female flowers. Gibberellin over-expression was deduced from the observed
phenotypic change, plants were taller and flowered earlier.
INTRODUCTION
Sex expression of cucumber plants can be effectively modified by exogenous
hormone application. Local changes in endogenous hormone levels are likely to
control reproductive organ development and sex phase succession in the plant.
Ethylene is considered the main feminizing hormone also in other cucurbits, while
gibberellin can induce male flowers mainly in cucumber (Perl-Treves and
Rajagopalan 2006). The AGII clone of ZYMV encodes a full length genome of the
potyvirus ZYMV, developed as an aphid-non-transmitted strain that infects various
cucurbit species, causing only mild symptoms (Arazi et al. 2001a). It can be used to
over-express foreign genes, inserted in-frame within the single polyprotein ORF of
the virus. This is achieved by infecting young cucurbit seedlings with the engineered
viral clone, taking advantage of the viral spread mechanism, and bypassing the need
of plant stable transformation and regeneration. We have cloned full open reading
frames of the ACC synthase gene from petunia, and the GA20 oxidase gene from
Arabidopsis, into AGII. Our purpose was to affect ethylene and gibberellin levels in
planta and assess the potential of this gene-expression tool to study sex modification
in cucurbits.
MATERIALS AND METHODS
Gene constructs
To construct the AGII-ACS expression vector, the petunia ACS3 coding region
(Papadopoulou et al. 2002; kindly provided by R. Woodson, Purdue University and
R. Grumet, Michigan State University) was amplified by PCR. Sense and antisense
primers contained PstI and a SalI sites at their 5' ends, respectively. To construct
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Cucurbitaceae 2008, Proceedings of the IXth EUCARPIA meeting on genetics and breeding
of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May 21-24th, 2008
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pAGII-GA, the AtGA20x1 (GA5) ORF was amplified from pCIB200 (Coles et al.
1999; kindly provided by P. Hedden, Rothamsted Research), by PCR with sense and
antisense primers, adding PstI and SalI sites to 5' and 3' ends of the GA5 sequence.
Amplified PCR fragments were digested and cloned into PstI/SalI sites of AGII
cDNA, to generate pAGII-ACS and pAGII-GA, respectively.
Plant growth and inoculation
Different cucumber genotypes (monoecious ‘Poinsett 76’, gynoecious line
‘Elem Female’, androecious line ‘Erez’) and squash (C. pepo L. 'Zucchini') were
grown under growth chamber conditions. Particle bombardment with a handheld
device, the HandGun (Gal-On et al. 1997), was used to deliver recombinant plasmids
into fully expanded cotyledons. For bombardment the tungsten stock solution was
mixed with equal volumes of cDNA in water and CaNO3 (1.25 M, pH 10.5) and 2 µl
per cotyledon were bombarded at pressure of 3 bar at a 2-3 cm distance. Symptoms of
infection in squash (vein clearing and mosaic) were visually determined. The
presence of viral RNA harboring the cloned insert was confirmed by RT-PCR. Total
RNA was extracted from leaf disks sampled from the non-inoculated ("systemic")
leaves of an infected plant, using TRI-reagent (Molecular Research Center, USA).
RT-PCR was conducted in a one-tube, single-step method with insert-flanking
primers (Arazi et al. 2001b).
Gas chromatography
Leaves were weighed and placed in a tightly sealed vial, incubated 2 h in the
dark, 3 ml of air were sampled to assay ethylene by GC using a Gas Chromatograph
Varian Aerograph model 3300.
RESULTS AND DISCUSSION
Gene constructs
We examined the effect of AGII constructs expressing enzymes for the
synthesis of two hormones, ethylene and gibberellin, on the plant phenotype and
hormonal balance. We designed primers that amplify the entire ORF of each gene,
providing the amino acids required as protease cleavage sites to release the foreign
protein from the viral polyprotein, as well as SalI and PstI restriction sites for inframe cloning. To avoid silencing, heterologous sequences were used. We first tested
our constructs in squash, because this species is infected more efficiently by ZYMV
compared to other cucurbits, and displays mild mosaic symptoms, facilitating visual
evaluation of infection. Confirmation of viral infection required RT-PCR analysis or
ELISA. The stability of the foreign gene insert in the viral RNA genome varies
among constructs (Arazi et al. 2001a). We monitored the integrity of the ACS insert
by RT-PCR in a time course experiment with six squash plants infected with the
ACS3 construct. We found that after 10 days all plants expressed an intact RNA
insert. After 14 days, smaller RNA species with deleted inserts appeared in some
plants in addition to the full-size molecules; at 20 dpi, the smaller species become
prevalent. Such instability represents a drawback of the system, providing only shortterm expression of some constructs.
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AGII-ACS increases ethylene production and could alter cucumber sex
expression
Infected squash plants expressing ACC synthase displayed very strong stunting
and produced small leaves (Fig. 1). The stunting syndrome was apparent also in
cucumbers of the gynoecious, monoecious and androecious sex types. Some of the
stunted plants recovered and resumed growth, possibly when the ACS insert
underwent deletion. Expression of ACC synthase caused a prominent increase in
ethylene production from excised leaves, compared to low, nearly undetectable levels
of control plants (Fig. 2). We did not observe changes in sex expression in squash. In
cucumber, recovering monoecious plants produced consecutive female flowers (Fig.
1B), not observed in control plants. However, these plants are developmentally
abnormal and milder expression would be required to perform physiologically
relevant sex expression studies.
A
B
Figure 1. A. Squash ACS3 expression. Right: plant expressing the AGII-ACS
construct. Left: control plants expressing insert-less AGII. B. Formation of female
flowers on monoecious cucumber expressing AGII-ACS.
GA20 oxidase expression
In squash, virus-mediated over-expression of GA20 oxidase resulted in prominent
elongation of internodes, leaf petioles and flower stems (Figs. 3 and 4A). In addition,
earlier and more profuse flowering was observed (Figs. 4B and C). There was a
typical gain of 2-3 days in anthesis time for male flowers, and a larger number of
flowers that developed in a given period; flowers were bigger and born on much
taller pedicels (~30 cm, compared to 15-20 cm in control plants). These are typical
gibberellin responses: in Arabidopsis, over-expression of GA20 oxidase resulted in
elongation and earlier flowering (Coles et al. 1999). Experiments are under way to
determine whether this construct will affect sex expression in cucumber.
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Ethylene (ppm/g x hr)
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0
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ACS over-expression
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Empty vector
Figure 2. Increased ethylene production by AGII-ACS squash plants, compared to
control plants expressing insert-less AGII.
Figure 3. Squash plants infected with the AG-GA strain are significantly taller. Left:
plants expressing GA20 strain, right: control plants transformed with insert-less AG
strain.
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A
Average internode length
Length (cm)
5.00
4.00
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3.00
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2.00
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1.00
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AGII-GA
control
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Internode number
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No. of flowers
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Rel. day of anthesis
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Figure 4. Increased internode length (A), flower number (B) and earlier flowering (C)
in squash plants expressing Arabidopsis GA20 oxidase. Asterisks denote significant
differences (two-tail distribution t-test, * - significant at p <0.01).
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